There are three general steps involved in the production of a protein utilizing genetic engineering techniques. The first step includes laboratory production of or isolation and purification of the desired gene which codes for a particular protein. The second step is the recombination of the gene with a proper transfer vector such as a plasmid. The third step includes transferring the recombined vector into a particular microorganism and inducing the microorganism to produce the particular gene product.
The present invention is directed towards a method of accomplishing the first step. Current methodology for in vitro production of genes by sequential addition of nucleotides consists of using chemical techniques (Itakura, K. and Riggs, A., Science 209:1401 (1980), (Khorana, H. G., Science 203:614 (1979), enzymatic procedures (S. Gillam, P. Jahnke, C. Astell, S. Phillips, C. A. Hutchinson, M. Smith, Nucleic Acids Res. 6,2973 (1979) and solid phase techniques to produce the desired DNA or RNA molecules. The chemical procedures of DNA synthesis are tedious because the reactions involved are non-specific such that extensive purification of the desired product is necessary after each operative step. Thus, synthesis of RNA and DNA by chemical techniques is significantly less desirable than enzymatic systems because greater amounts of starting material are required to produce comparable yields thereby increasing costs, the purification techniques are both time consuming and wasteful where some product is lost at each step, and the large number of steps involved in blocking and unblocking reactive sites provide many opportunities for error and risk of contamination by degradative enzymes which destroy the synthesized product.
Enzymatic synthesis of RNA using polynucleotide phosphorylase and T4 RNA ligase have been reported. The polynucleotide phosphorylase method is limited to production of oligodeoxynucleotides. Under controlled conditions, polynucleotide phosphorylase adds predominantly a single nucleotide to a short oligodeoxynucleotide which is then isolated by chromatographic techniques solid phase techniques are performed by binding the nucleotide chain to a solid support material and using the above chemical methodology to add nucleotides stepwise.
The present invention teaches the composition and method of production of a compound useful in the process of synthesizing a predetermined sequence of RNA using the enzyme T4 RNA ligase. Processes for transcribing the synthesized RNA into DNA so that the synthesized gene may be incorporated into a plasmid, inserted into a microorganism and expressed are known in the art.
The existence, purification and mechanism of the enzyme T4 RNA ligase which is isolated from Escherichia coli infected with bacteriophage T4 have been described (Silber, R., Malathi, V. G. & Hurwitz, J., Proc. Natl. Acad. Sci. (1972) 69:3009). This enzyme catalyzes the formation of a phosphodiester bond between the phosphate group on the 5' end of the donor nucleotide and the hydroxyl group on the 3' end of the recipient oligonucleotide as shown below. ##STR1## Japanese Patent Application No. 1980-19003 (published Feb. 9, 1980 teaches the utilization of T4 RNA ligase to extend a polynucleotide by adding a single mononucleoside diphosphates (pNp) onto the 3'end of a nucleotide sequence with no terminal phosphate groups. This methodology requires as the starting substrate a trinucleotide, which must be either commercially available or synthesized by chemical means, and requires adenosine triphosphate (ATP) as an energy source for the reaction. Additionally, the invention teaches only the addition of a single base and is not capable of producing oligo or polynucleotides for which sequential addition is required. EQU HOApApAOH+pCp+ATP.fwdarw.HOApApApCp+AMP+PPi
Abbreviations to be used in this application for convenience are provided in Table 1.
TABLE 1 ______________________________________ Abbreviation Definition ______________________________________ A(dA) Adenosine (deoxyadenosine) C(dC) Cytidine (deoxycytidine) G(dG) Guanosine (deoxyguanosine) U(dU) Uridine (deoxyuridine) I(dI) Inosine (deoxyinosine) T Thymidine N,X,Y,Z Any ribonucleotide or deoxyribonucleotide DNA Deoxyribonucleic acid RNA Ribonucleic acid BAP Bacterial Alkaline Phosphatase VPD Venom phosphodiesterase AppNp A.sup.5 'p.sup.5 'pN.sup.3 'p (see text for further explanation) &gt;P 2'-3' cyclized phosphodiester bond ul Microliter nm Nanometer TEAB Triethylammonium Bicarbonate Buffer pA Adenosine-5'-monophosphate ______________________________________
The standard form of representing 5' phosphate groups of a nucleotide as preceding the nucleotide abbreviation and the 3' phosphate group as succeeding the nucleotide abbreviation is used herein.